Animals

Experiments were carried out on adult female Sprague-Dawley rats (N = 31, weighing between 225 and 250 g, Charles River Laboratories, Saint-Constant, QC, Canada). Rats were housed in groups of three under controlled conditions of temperature (21 ± 1 °C), humidity (55%), and lighting (12-hour light/dark cycle, with lights turned on at 07:00). Rats had unrestricted access to food and water. Before the experiments, rats were left undisturbed for a minimum of 5 days to acclimate to their housing environment. All procedures were granted approval by the Animal Care Committee (Animal Use Protocol #2017–7922) at the Montreal Neurological Institute-Hospital (The Neuro), following guidelines established by the Canadian Council on Animal Care.

Induction of hemi-parkinsonism

Hemi-parkinsonism was induced by unilateral injection of 6-OHDA, as previously described (Frouni et al. 2019, 2022; Hamadjida et al. 2020). Initially, animals were administered desipramine (10 mg/kg subcutaneously [s.c.], MilliporeSigma, Oakville, ON, Canada) and pargyline (5 mg/kg s.c., MilliporeSigma) to prevent noradrenergic neuron damage (Ungerstedt 1968). Rats were anaesthetised using isoflurane (2–4%) in 100% oxygen (1 L/min) and positioned in a stereotaxic frame (David Kopf Instruments, Tujunga, CA, USA). After a period of 30 min, a unilateral injection of 6-OHDA hydrobromide (7 µg/µL, MilliporeSigma) was performed in the right medial forebrain bundle and the injection site coordinates relative to Bregma were as follows: antero-posterior: −2.8 mm, medio-lateral: −2.0 mm, dorso-ventral: −9.0 mm (Paxinos and Watson 2007). The incisor bar was set 3.3 mm below the ear bars. For comparison, sham-lesioned animals received an injection of 6-OHDA vehicle (0.9% saline with 0.02% ascorbate) using the same coordinates mentioned above.

Assessment of hemi-parkinsonism

After a three-week recovery period, animals underwent the cylinder test to evaluate the extent of hemi-parkinsonism, by counting the number of rears of both forepaws, separately and concomitantly, on a cylinder wall (Schallert et al. 2000; Frouni et al. 2022). Rats were placed in a transparent cylinder (14 cm diameter × 28 cm height). Rats were recorded for a duration of 10 min and their behaviour was analysed post hoc. Only animals that used the forepaw ipsilateral to the lesion side (the non-parkinsonian forepaw) in at least 70% of rearing were chosen for further investigation. This criterion represents a score indicative of a striatal dopamine deficit greater than 88% (Schallert et al. 2000).

Experimental groups

Rats that met the inclusion threshold after the cylinder test were divided into four distinct groups: (1) sham group (vehicle-lesioned, treated with vehicle [0.1% ascorbate in 0.9% NaCl], N = 8), (2) parkinsonian group (6-OHDA-lesioned, treated with vehicle, i.e. not exposed to L-DOPA, N = 8), (3) mildly dyskinetic group (6-OHDA-lesioned, treated with L-DOPA and exhibiting mild dyskinesia, N = 7), (4) severely dyskinetic group (6-OHDA-lesioned, treated with L-DOPA and exhibiting severe dyskinesia, N = 9). Vehicle and L-DOPA treatments were administered once daily s.c. for 14 days. L-DOPA was administered as L-DOPA/benserazide 10/15 mg/kg.

Assessment of abnormal involuntary movements severity

After the 14-day dyskinesia induction period, L-DOPA-treated 6-OHDA-lesioned rats were injected with L-DOPA (10/15 mg/kg, s.c.) and the severity of axial torsion, limb movements, oro-lingual stereotypies (ALO) abnormal involuntary movements (AIMs) was evaluated by an experienced observer blinded to the treatment, according to a scale that evaluates severity-based, i.e. “amplitude” and time-based, i.e. “duration” AIMs (Cenci and Lundblad 2007). ALO AIMs were assessed for a duration of 2 min every 20 min, for a total observation period of 180 min, plus a baseline assessment. In summary, one experimental session lasted 3 h. During these 3 h, we assess the severity of AIMs every 20 min. Each of these assessments last 2 min. The duration and amplitude of the individual ALO components were individually rated on a scale ranging from 0 to 4 for each monitoring interval, with a maximum obtainable score of 36 per session. The “integrated” ALO AIMs score was defined as the product of ALO AIMs duration × ALO AIMs amplitude, as previously described (Frouni et al. 2018).

Tissue preparation

Animals were administered their regular L-DOPA or vehicle treatment and, 45 min later, were euthanised by isoflurane overdose (2–4%) and subjected to trans-cardial perfusion with ice-cold 0.9% saline. Brains were collected and flash-frozen in isopentane at -56 °C and stored at -80 °C until cryostat sectioning. Brains were set in optimal cutting temperature (OCT) in a cryostat at -20 °C (Leica CM3050 S; Leica Microsystems, Richmond Hill, ON, Canada) and sliced coronally into 12-µm thick sections. Sections were thaw-mounted on SuperFrost® Plus slides (Thermo Fisher Scientific, Mississauga, ON, Canada) and stored at -80 °C until use.

Immunohistochemistry

Extent of lesion on striatal brain sections was examined by immunohistochemistry using a mouse monoclonal antibody raised against tyrosine hydroxylase (TH) (1:1000, MilliporeSigma, #MAB318) as previously described (Kwan et al. 2022). Frozen striatal brain sections mounted on slides were air-dried overnight at room temperature and then fixed by immersing them in pre-cooled acetone (-20 °C) for 10 minutes, followed by a 20-minute air-drying period. Sections were immersed in 0.5% H2O2 for 10 minutes to block endogenous peroxidase activity. Thereafter, sections were incubated for 1 h in 10% normal goat serum (NGS) and 5% bovine serum albumin (BSA) in Tris buffered saline (TBS; 100 mM Tris-Cl, pH 7.40, containing 240 mM NaCl) containing 0.3% Triton X-100 to block non-specific immunoreaction. Then, sections were incubated with the TH antibody in 5% NGS and 2% BSA in TBS containing 0.1% Triton X-100 (TBS-T) overnight at 4 °C. Sections were incubated in the presence of goat anti-mouse biotinylated secondary antibody (1:200, Invitrogen, Waltham, MA, USA, #31,800) in 5% NGS and 2% BSA in TBS-T for 1 h, followed by an incubation in avidin-biotin complex detection kit (ABC; Vector Laboratories, Newark, CA, USA, #PK-6100) for 2 h. The immunoreaction in sections was visualised in TBS-T containing 1.25 mg/mL nickel ammonium sulphate hexahydrate (MilliporeSigma, #574,988), 0.25 mg/mL 3,3’-diaminobenzidine (MilliporeSigma, #D5637) and 0.015% H2O2. Finally, sections were dried, rehydrated in water, dehydrated in graded alcohol solutions, cleared with xylene and coverslipped using Permount mounting medium (Thermo Fisher Scientific, SP15-100).

TH-immunoreactivity levels were assessed using densitometry in sections encompassing the dorsolateral striatum (Bregma ∼ +1.20 mm). Images were captured by a Nikon Eclipse E800 microscope (The Neuro Microscope Core Facility) using Stereo Investigator software (MBF Bioscience, Williston, VT, USA, version 11). The optical density in both ipsilateral and contralateral dorsolateral striata was measured across four adjacent brain sections using ImageJ software (NIH, Bethesda, MD, USA, version 1.53c). The average relative TH optical density was computed for each side, on every animal.

[3H]-NFPS autoradiographic binding

As mentioned in the Introduction, the regions of interest were selected for analysis based on their implication in the motor loop of the basal ganglia (McGregor and Nelson 2019) and were identified from a standard rat brain atlas (Paxinos and Watson 2007). The investigated areas consist of the primary motor cortex (M1), striatum (encompassing both the caudate nucleus and putamen, henceforth abbreviated as CPu), globus pallidus (GP), entopeduncular nucleus (EPN), subthalamic nucleus (STN), ventral anterior/ ventral lateral (VA/VL) nuclei of the thalamus, as well as the SN.

Four consecutive sections were processed for each brain area to determine total binding, and four to evaluate non-specific binding. Sections were thawed and dried at room temperature overnight. Sections were pre-incubated by washing twice in Krebs-HEPES buffer (50 mM, pH 7.4) for 15 min at room temperature and then incubated in Krebs-HEPES binding buffer (50 mM, pH 7.4) containing 10 nM [3H]-NFPS (American Radiolabeled Chemicals, St. Louis, MO, USA; specific activity: 59.72–60.23 Ci/mmol) for 60 min at room temperature to assess total binding. Non-specific binding was assessed by the addition of 1 µM Org-25,935 (Cedarlane Laboratories, Burlington, ON, Canada) to the Krebs-HEPES binding buffer containing 10 nM [3H]-NFPS. Sections were then washed three times in 4 ºC Krebs-HEPES buffer (50 mM, pH 7.4; 4ºC) for 10 min to end the binding. Following this, sections were briefly dipped in 4 ºC ddH2O and air dried at room temperature. Sections were opposed to [3H]-sensitive Biomax MR films (MilliporeSigma, Canada) for 3 weeks at room temperature, along with [3H]-microscale standards (ART0123B and ART0123C, 5 mm × 7 mm; American Radiolabeled Chemicals). Films were developed and autoradiograms were obtained for densitometric analysis.

Autoradiograms were analysed using optical densitometry with ImageJ software (version 1.52p). Beta-emitting [3H]-microscale standards of known radioactivity were employed to construct a reference curve that correlates radioactivity with the grey scale values observed in autoradiograms (Zilles et al. 2002). This curve was then used to quantify the intensity of the signal in nanoCuries (nCi) per mg of tissue. Background values were subtracted from total and non-specific binding values. Specific binding was calculated by subtracting non-specific binding values from total binding.

Statistical analysis

Cylinder test data are presented as the mean ± standard error of the mean (SEM) and were analysed by one-way analysis of variance (ANOVA) followed by Tukey’s tests. Relative striatal TH optical density expression is presented as a percentage of the unlesioned hemisphere and were analysed using Student’s t test.

Time courses of ALO AIMs scores are presented as the median and were analysed by computing the area under the curve (AUC), followed by unpaired Welch’s unequal variances t-test.

For each region of interest, specific [3H]-NFPS binding levels are displayed as the mean ± SEM. Levels of binding for each region of each animal group were analysed by planned comparisons with multiple Student’s t tests and P values were corrected using the Holm-Sidak multiple comparisons test.

Correlation between PD severity and ALO AIMs scores with specific [3H]-NFPS binding were determined using the Pearson correlation coefficient.

For all analyses, statistical significance was assigned when P ˂ 0.05. Statistical analyses were performed with GraphPad Prism 8.4.3 (GraphPad Software, Boston, MA, USA).